The present invention relates generally to the field of enhancing the repair of wounds in mammalian tissue, e.g., following injury, burn, surgery and skin grafting or tissue transplantation, and inducing neovascularization therein.
Wound repair and tissue generation in normal and impaired wound healing conditions is a major focus in medicine. In particular, the capability to achieve wound healing or develop tissue growth in an impaired wound healing, environment remains a problem. The mechanisms of normal wound healing, hypertrophic and keloid scarring, as well as the generation of new tissue growth have been postulated to be related, particularly at the growth factor level. The potential for growth factors to enhance wound healing, soft tissue generation, scar manipulation, and tumor activity has stimulated intense investigative efforts over the past few years. Unfortunately, these studies have yet to provide a clinically effective delivery system or clinically significant results in human skin.
Experimental evidence suggests that human vascular endothelial growth factor (VEGF), for example, has an important function in the maintenance of the vasculature in healthy tissues, in wound healing and in angiogenesis. VEGF is a potent mitogen for endothelial cells, and causes cytoplasmic accumulation of calcium, changes in cell morphology, an increase in cell division, and altered gene expression (up-regulates proteases). VEGF also inhibits the maturation of dendritic cells, permeabilizes vascular beds (VEGF enhances vesicular-vacuolar organelles) and is responsible for the autocrine growth of AIDS-Kaposi sarcoma cells.
At low levels, VEGF is expressed by a variety of normal cells including keratinocytes [C. P. Kiritsy and S. E. Lynch, 1993, Crit. Rev. Oral Biol. Bed., 4:729-760] and macrophages in the healing of cutaneous wounds. VEGF is also found in the endometrium and corpus luteum; is produced by podocytes or renal glomerulus, by prostatic epithelium and by epithelial cells of the adrenal cortex and lung. VEGF is upregulated during wound healing, psoriasis and delayed type hypersensitivity. It is produced by cardiac myocytes in ischemic myocardium and by synovial lining cells in the pannus of rheumatoid arthritis. It is constitutive in many tumors, such as tumors of the colon, stomach, pancreas, kidney, bladder, breast and glioblastoma. Most malignant cells, including melanoma cells, express VEGF. Its expression can be induced by other growth factors, such as transforming growth factor (TGF-xcex2) and platelet derived growth factor (PDGF) and cytokines, or by hypoxic environmental conditions [S. A. Eming el al, 1995, J. Invest. Dermatol., 105:756-763]. Its over expression leads to hypervascularization which is often associated with chronic inflammatory diseases and cancer [see, e.g., F. Grinnel, 1992, J. Cell Sci., 101:1-5, V. Falanga el al, 1994, J. Invest. Dermatol., 102:125-127; G. F. Pierce et al, 1991, J. Cell. Biol., 45:319-326].
A variety of VEGF constructs and uses in neovascularization and wound healing have been proposed. For example, see International Patent Application WO96/26736, published Sep. 6, 1996, which relates to VEGF-B proteins useful to accelerate angiogenesis in wound healing, International Patent Application WO95/24473, published Sep. 14, 1995, relates to VEGF-xcex2polypeptide, useful for wound healing and periodontal disease; European Patent Application No. 550296, published Jul. 7, 1993 relates to VEGF protein, used for promoting angiogenesis in treatment of cardiac angiopathy, wounds, burn injuries, postoperative tissue damage, etc.; U.S. Pat. No. 5,219,739, issued Jun. 15, 1993, relating to DNA sequences, vectors and transformed cells for producing VEGF for treating wounds European Patent Application No. 506477, published Sep. 30, 1992, relating to VEGF sub-units for inducing tissue repair and growth; European Patent Application No. 476983, published Mar. 25, 1992, and relating to VEGF 11 for coating blood vessels or to promote tissue repair; U.S. Pat. No. 5,073,492, issued Dec. 17, 1991; and U.S. Pat. No. 5,194,596, issued Mar. 16, 1993, among others.
The use of replication deficient adenovirus vectors (Ad) to deliver VEGF to heart, smooth muscle and endothelial cells, as well as delivery of such vectors via subcutaneous injection has been the subject of much experimentation. For example, replication defective adenoviruses carrying VEGF genes have been described in, e.g., J. Muehihauser et al, 1995, Circul. Res., 77(6):1077-1086 and J. Muhlhauser et al, 1994, J. Cell. Biochem., Supp. 0 (18 Part A), p. 239. The Muhlhauser references refer to the delivery of a replication deficient adenovirus carrying a human VEGF-165 gene under the control of the cytomegalovirus promoter to human umbilical vein endothelial cells and rat aorta smooth muscle cells. The same vector was injected subcutaneously in mice, and two weeks post injection, histological evidence of neovascularization in the tissue surrounding the site of injection was observed. Similarly, C. J. Magovern et al, 1996, Annals Thor. Sure., 62(2):425-434 refers to the use of a replication defective Ad carrying the gene for VEGF in direct myocardial injection to accomplish gene transfer. These authors noted sustained and localized expression of VEGF for up to 7 days after a single injection, and posited that this strategy may be used to stimulate angiogenesis in ischemic myocardium [see, also, R. Ziegelstein et a/, 1994, Circul., 90(4), part II, p. 1899].
The principal growth factor found in platelets as well as macrophages, fibroblasts and endothelial cells is platelet derived growth factor (PDGF), a 30 Kda protein dimer existing in three different isoforms (PDGF-AA, PDGF-AB and PDGF-BB), which is released at a site of injury within the body. The mitogenic effects of PDGF-BB isoform, which can bind to the PDGF-xcex2 receptor, on fibroblasts, smooth muscle cells and other mesenchymal cells have been extensively documented. The chemoattractive effects mediated through the PDGFB receptor have implicated PDGF-BB and PDGF-AB in the physiologic process of wound healing and tissue repair and the pathologic process of atherosclerosis.
PDGF-BB has been shown to be an integral part of the initial and early stages of wound healing, with its participation in the inflammation and granulation tissue stages respectively. PDGF-BB was also shown to be present in wounds and also within the blister fluid of burn patients. The final stage of wound healing, in which scar tissue remodelling occurs, can largely be attributed to the autocrine production of PDGF-BB from dermal fibroblasts. Such autocrine loops are initially stimulated in a paracrine fashion with platelet PDGF, which ultimately results in the deposition of extracellular molecules such as fibronectin and tenascin. Chondroitin sulfate may also be deposited which may inhibit the action of collagenase. In wound healing it has also been shown that there is also a major reorganization of collagen types I and III. The accumulation of such molecules in connective tissue is associated with diseases such as rheumatoid arthritis and atherosclerosis. This may implicate the involvement of PDGF in the pathogenesis of these diseases. The sequence homology of PDGF-B with a viral oncogene from Simian Sarcoma virus (v-sis), also implicates PDGF-B in normal and neoplastic development, with many tumors being intimately involved with the production of PDGF-BB.
Systematic analyses of the roles of individual growth factors and cytokines for their angiogenic properties have been difficult in disease-related conditions because experimental over expression or suppression of expression of growth factors in human cells could not be achieved consistently under in vivo conditions. Indirect evidence of the potential roles of growth factors came from expression studies in diseased skin in which mRNA or protein levels were determined.
Application of exogenous growth factors to wounds, for example, has shown efficacy in wound healing in some experimental animal models, e.g., topically applied in impaired wounds, but the few clinical studies are difficult to interpret [Kiritsy, cited above], apparently due to the limited biological half life of the growth factors when applied topically or the difficulty of obtaining frequent biopsies for analyses. Only minimal improvement in non-impaired wounds has been shown. For example, PDGF-BB has been applied to chronic wounds such as diabetic ulceration. The efficient penetration of PDGF to cells in the wound bed and a demonstrable healing effect has been difficult to achieve and may be enhanced by wound dressings [Pierce, G. F, et al, 1995 J. Clin. Invest., 96:1336-1350, Ono, I., et al, 1995 J. Dermatol. Sci., 10:241-245]. Up-regulation of proteinases in non-healing wounds may contribute to the rapid degradation of topically applied PDGF [Yager, D. R., et al, 1996 J. Invest. Dermatol. 107:743-748]. While exogenous PDGF-B can enhance acute experimental wounds in animals [see, e.g., Lynch, S. E., et al. 1987, Proc. Natl. Acad. Sci. USA 84:7696-7700; Pierce, G. F., et al 1991 Am. J. Pathol. 138:629-646; Pierce, G. F., 1992, Am. J. Pathol. 140:1375-1388], results in human trials of chronic wounds have been less effective [Steed, D. L. et al, 1992, Diabetes Care. 15:1598-1604; Robson, M. C., et al, 1992, Annals Plastic Surg. 29:193-201], necessitating improved methods for drug delivery. One alternative approach is to express the PDGF gene in the fibroblasts of wounds. Due to the oncogenic potential of the PDGF gene [Gazit, A.,. et al., 1984 Cell 39:89-97] such procedures require a strictly circumscribed expression of PDGF.
A major stumbling block to the use of growth factor therapy for the treatment of non-healing wounds has been the difficulty in protein delivery [Elia and Friend, 1975, J. Cell Biol., 65:180-191; Chen and Okayama, 1989, Mol. Cell. Biol., 922:2745-2752; D. Greenhalgh et al., 1990, Am. J. Pathol., 136:1235-1246; T. Mustoe et al, 1994, Arch. Sur2., 129:213-219; G. Hubner et al., 1995, Cytokine, 8:548-556]. All animal and clinical trials to date have thus far required large doses and repeat administration of growth factor in order to favorably influence wound healing (D. Greenhalgh et al., cited above, T Mustoe et al., cited above) Previously, methods including in vivo retroviral transfection, in vitro DNA transfection and autologous transplantation, plasmid DNA, and DNA-coated particle bombardment, have met with limited success because of poor gene transfer and limited biologic effect [Chen and Okayama, cited above; C. Andree et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 21188-12192; G Kruegger et al., 1994, J. Invest. Dermatol., 103: 76-84s; U. Hengge et al., 1995, Nature Genet., 10:161-166; S. Benn et al., 1996, J. Clin. Invest., 98:2894-2902; I. Ciernik et al., 1996, Human Gene Ther., 7:893-899].
Thus, there remains a need in the art for methods and compositions useful in treating wounds, as well as in inducing cell growth, neovascularization and repair in injured or grafted mammalian, particularly human, tissue.
In one aspect, the present invention involves a method for increasing/inducing vascular development in mammalian tissue by delivering to the tissue a replication defective recombinant virus, preferably adenovirus, comprising a human growth factor gene under the control of regulatory sequences capable of directing expression of that gene in the tissue. The tissue can include the heart, arteries, veins or organs, such as kidney, liver, etc. In a selected embodiment, the tissue is human skin. This method enables the production of growth factors by different cells in the skin and/or in other tissue.
In another aspect, the invention provides a method for enhancing the repair of a wound in a mammal comprising administering to the mammal (preferably a human) a recombinant replication defective virus, preferably adenovirus, comprising a growth factor gene under operative control of regulatory sequences which direct the expression of said growth factor in the area of said wound. Expression of the growth factor thus enhances fibroblast growth and formation into a matrix, enhances keratinocyte growth and angiogenesis, and thus enhances wound closure.
Another aspect of this invention is a method for administering into poorly vascularized tissue (eg., skin) to be transplanted just prior to transplantation the virus containing the growth factor gene. Specifically, the genes desirably administered to a skin culture are VEGF121 and PDGF-B.
In still another aspect, the invention provides a composition comprising tissue to be transplanted (e.g., human skin) which is infected with a recombinant virus which directs expression of VEGF or PDGF prior to transplantation.
In still another aspect, the invention provides a method of engrafting tissue onto a site of tissue injury in a mammal by (a) infecting a culture of human fibroblasts with about 10 pfu/cell of a recombinant replication defective virus, preferably adenovirus, comprising a selected growth factor gene under operative control of regulatory sequences which direct the expression of said gene product in said fibroblasts prior to transplantation; (b) placing said infected fibroblasts onto the site, and (c) placing tissue to be grafted at the site over the infected fibroblasts. The tissue (a) may also be treated with the virus.
In yet a further aspect, the invention provides a novel animal model for disease comprising an immunodeficient mouse, e.g., SCID or RAG mouse, stably engrafted with mammalian adult skin, preferably human skin, or other mature tissue infected with a recombinant virus which directs expression of VEGF or PDGF prior to engraftment.
In still a further aspect, the invention provides a method for screening angiostatic compounds useful in the treatment of pathological states such as VEGF-induced hemagiomas, particularly those resistant to conventional treatment, and other pathological states, such as cancers. This method comprises the steps of exposing the human skin graft on the animal model described above (in which the human tissue or at least a fibroblast layer was infected with a recombinant adenovirus expressing VEGF) with a test compound and observing the effect of the test compound on the process of formation and resolution of hemangiomas or growth of fibroblasts, keratinocytes, and/or angiogenesis in the graft. Compounds which reduced the process of hemangioma formation or enhanced the resolution thereof or enhanced cell grwoth and/or angiogenesis may be selected for the treatment of such hemangiomas in humans.
Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof.